1. Field of the Invention:
This invention relates to a process for preparing monosialganglioside.
A ganglioside is a member of a class of glycosphingolipids found abundantly in human and animal brains and is represented by the following general formula: ##STR1## wherein GalNAc means N-acetylgalactosamine, Gal denotes galactose, Glc stands for glucose, NeuNAc is N-acetyl- and/or N-glycolylneuraminic acid (hereinafter called simply "sialic acid"), Cer denotes ceramide, n means an integer from 0-3, and m stands for an integer from 1-3. Depending on the number and bound position or positions of sialic acid residue or residues which are one of its components, there are many molecular species. Therefore, the term "ganglioside" as used herein is a general designation for these compounds.
Ganglioside may be roughly classified, depending on the number of sialic acid residue or residues bound per molecule, into monosialoganglioside (GM), disialoganglioside (GD), trisialoganglioside (GT), and tetrasialoganglioside (GQ) in which four sialic acid residues are bound. They can be classified further depending on the position or positions of the sialic acid residue or residues bound. The following molecular species of ganglioside are known: GM.sub.1 [n=0, m=1 in the general formula (1)] as GM, GD.sub.1a (n=1, m=1) and GD.sub.1b (n=o, m=2) as GD, GT.sub.1b (n=1, m=2) as GT, and GQ.sub.1b (n=2, m=2) as GQ, etc.
The subscript of GM.sub.1 means that all of the four saccharides of the basic oligosaccharide are present. The oligosaccharide formed by elimination of the end Gal from GM.sub.1 is called "GM.sub.2 ", and the oligosaccharide obtained by further elimination of GalNAc is called "GM.sub.3 ".
The saccharide formed by complete elimination of the sialic acid residue is called asialo GM.sub.1 or GA.sub.1.
The action of ganglioside in living bodies has been being elucidated in recent years. It has been reported that GM.sub.1 is effective for the repair and treatment of disorders of central and peripheral nervous systems [for example, Acta Neuropathologica, 62, 46-50 (1983); Agnati, L. F., et al., Acta Physiolgica Scandinavica, 119, 347-363 (1983)].
In Italy, a GM.sub.1 -containing ganglioside mixture with the name of "Cronacial.RTM." is marketed as a therapeutic drug for peripheral nerve diseases. An application for patent has been filed on this mixture (now Japanese Patent Laid-Open No. 34912/1977).
2. Description of the Prior Art:
As a conventional process for the formation of GM.sub.1 from a ganglioside species other than GM.sub.1, it has been known to cause neuraminidase, a desialidase, to act on the ganglioside species [for example, Richard Kuhn, et al., Chemische Berichte, 96, 866 (1963)].
It has also been reported that GM.sub.1 was formed without using any enzyme [S. Ando, et al., The Journal of Biological Chemistry, 254(23). 12224-12229 (1979)]. Namely, it has been reported that a mixture of GT.sub.1a, GT.sub.1b, GD.sub.1b, GD.sub.1a and GM.sub.1 was obtained by holding GQ at pH 3 and 80.degree. C. for 30 minutes in an aqueous solution of formic acid. According to the thin-layer chromatogram in this report, the proportion of GM.sub.1 in the thus-obtained mixture is however extremely small and a further confirmation is hence necessary as to the formation of GM.sub.1.
It has also been known that the sialic acid residue or residues of ganglioside are eliminated by acidolysis [for example, E. Klenk, Hoppe-Seyler's -Zeitschrift fur Physiologische Chemie, 270, 185(1941); and L. Svennerholm, et al., The Journal of Biological Chemistry, 248, 740(1973)].
These prior art processes are intended to eliminate all sialic acid and the reaction products are GA.sub.1 and neutral glycosphingolipids formed by elimination of 1-3 saccharides from the basic oligosaccharide. They are not intended to form GM.sub.1.
As ganglioside supply sources employed presently in the industry, brains of bovine, swine and the like are used. Ganglioside available from these brains generally contains molecular species of ganglioside other than GM.sub.1 in higher proportions than GM.sub.1. The present inventors obtained ganglioside in a purified form by a process known per se in the art, namely, by dehydrating bovine brain with acetone, extracting it with a mixed solvent of chloroform, methanol and water by the process proposed by Svennerholm, et al. [Biochemical et Biophysica Acta, 617, 97-109(1980)], converting the extract into an aqueous solution of ganglioside by the Folch's distribution [J. Folch, The Journal of Biological Chemistry", 226, 497-509 (1957)], purifying it by DEAE-Sephadex-A25 [R. W. Ledeen, et al., Journal of Neurochemistry, 21, 829 (1973)], followed by a silica gel column fractionation. The composition of the above-purified ganglioside was 14% of GM.sub.1, 45% of GD.sub.1a, 10% of GD.sub.1b, 22% of GT.sub.1b and 2% of GQ.sub.1b.
In the above-described ganglioside preparation, "Cronacial.RTM.", the content of GM.sub.1 is 21% according to the literature for the preparation. If the molecular species of ganglioside other than GM.sub.1 in the ganglioside mixture can be converted to GM.sub.1, the effective component can be obtained in a correspondingly higher proportion. Although GM.sub.1 can be formed by causing neuraminidase to act on ganglioside, this enzyme is expensive and therefore this process does not appear to be practical process from the economical standpoint.